This invention relates to a process for producing a desulfated polysaccharide, in which a sulfate group(s) bonded to a primary hydroxyl group(s) of a sulfated polysaccharide is/are selectively desulfated, and a desulfated heparin obtained by this production process.
In order to provide a sulfated polysaccharide having an biological activity., various methods for desulfating a sulfated polysaccharide have been studied. As a method for desulfating a sulfated polysaccharide, there has been known a method of carrying out desulfation by an acid catalyst in hydrogen chloride/methanol (Kantor T. G. and Schubert M., J. Amer. Chem. Soc. Vol. 79, p. 152 (1957)). However, in this method, it is impossible to desulfate only a specific sulfate group(s), and cleavage of a sugar chain due to methanolysis of a glycoside bond occurs to lower the molecular weight, whereby the yield of a reaction product having an original chain length is lowered.
As a method for carrying out desulfation with good yield, there may be mentioned solvolysis carried out in an aprotic solvent such as dimethylsulfoxide (DMSO), N,N-dimethyl-formamide (DMF) or pyridine, etc. (Usov A. et al. Carbo-hydr. Res., Vol. 18, p. 336 (1971)) or in DMSO containing a small amount of water or methanol (Nagasawa K. et al. Carbohydr. Res., Vol. 58, p. 47 (1977), Nagasawa K. et al. J. Biochem., Vol. 86, p. 1323 (1979)). It has been known that the reaction mechanism of solvolysis is a reverse reaction of a sulfation reaction carried out by using a complex of sulfur trioxide and an amine in an aprotic solvent. This reaction can be used as a method for selective desulfation of an N-sulfate group(s) by controlling reaction conditions. However, an O-sulfate group(s) bonded to a primary or secondary hydroxyl group(s) cannot be eliminated. Further, when this method is applied to an oligosaccharide or a polysaccharide, there are problems that an operation of removing the solvent such as DMSO, etc. after the reaction is complicated, it is necessary to raise the reaction temperature in order to carry out complete desulfation, cleavage of a glycoside bond occurs under such reaction conditions, etc.
On the other hand, as a method for specifically desulfating a sulfate group(s) bonded to a primary hydroxyl group(s) of a saccharide, there may be mentioned a method of using N,O-bis(trimethylsilyl)acetamide (BTSA) (Matsuo, M. et al., Carbohydr. Res., Vol. 241, pp. 209-215 (1993)). When this method is applied to heparin, a sulfate group at 6-position of glucosamine is removed relatively specifically. However, as a result of examining a detailed structure thereof by an enzymatic digestion method, it has been found that simultaneously with eliminating a sulfate group(s) bonded to a primary hydroxyl group(s), elimination of a small amount of an N-sulfate group(s) occurs. Therefore, a method for removing a sulfate group(s) bonded to a primary hydroxyl group(s) with higher selectivity has been demanded. Development of a method for specific elimination of a sulfate group(s) bonded to a primary hydroxyl group(s) is very important for providing a sulfated polysaccharide for the purpose of creating a medicine having a preferred biological activity to human. For example, dextran sulfate, xylan sulfate, chondroitin sulfate, heparin, etc. which are sulfated polysaccharides have been used as a lipometabolism-improving agent or an anti-thrombus agent. However, it has been also known that in one in which sulfate groups are introduced artificially, the positions of introduced sulfate groups cannot be specified, and accompanied with introducing a large amount of sulfate groups, a side effect that a tendency of hemorrhage from tissues is strengthened is caused. Also, sulfated polysaccharides derived from natural substances are different in the positions and amount of sulfate groups, respectively, depending on difference in origin, and the physiological activities of the respective sulfated polysaccharides are also different slightly.
Desulfation of heparin having ability of specifically bonding to various physiological active proteins is considered to be extremely important. For example, in the structure of heparin which interacts with a basic fibro-blast growth factor (bFGF) to accelerate its stabilization and activity to cellular proliferation, N-sulfate groups and sulfate groups at 2-position of iduronic acid are contained abundantly, and no sulfate group at 6-position is required (Ishihara, M. et al., Glycobiology, Vol. 4, pp. 451-458 (1994)). On the other hand, in order to accelerate an activity to an acidic fibroblast growth factor (aFGF) or FGF-4 (Kaposi""s sarcoma FGF), abundant sulfate group at 6-position is also required (Ishihara, M., Glycobiology, Vol. 4, pp. 817-824 (1994)). Therefore, in a heparin selectively desulfated at 6-position, obtained by removing a sulfate group(s) of a primary hydroxyl group(s) (hydroxyl group at 6-position) of glucosamine from heparin, an anticoagulant activity is lowered as compared with heparin before desulfation is carried out. However, an effect of specifically accelerating a bFGF activity is maintained and also an effect of suppressing undesired physiological activities generated from interactions with a large number of other physiological active molecules in vivo at a low level can be expected.
As a composition using a fibroblast growth factor (FGF) and polysaccharides, there have been proposed, for example, a medical composition comprising FGF, a sulfated polysaccharide having an antiviral activity and an excipient described in Japanese Provisional Patent Publication No. 80583/1994, a composition comprising FGF, 2-0-sulfated L-iduronic acid and N-sulfo-D-glucosamine and also containing at least one of oligosaccharides having ability of bonding to FGF constituted by 8-18 saccharides described in European Patent Publication No. 509517, and a complex of FGF mutein and glycosaminoglycan or a composition into which these are formulated described in Japanese Provisional Patent Publication No. 40399/1990. Among them, as the sulfated polysaccharide used in the medical composition described in U.S. Pat. No. 5,288,704, desulfated one is not described, and the medical composition is aimed at enhancement of an antiviral activity by a cooperative action. Also, the composition described in European Patent Publication No. 509517 uses a specific oligosaccharide which is not desulfated, and the composition described in Japanese Provisional Patent Publication No. 40399/1990 uses natural glycosaminoglycan which is not subjected to desulfation treatment.
For the purpose of making an inherent biological activity of the sulfated polysaccharide being expressed more specifically with less side effects such as a hemorrhagic action, etc. by desulfating only a sulfate group(s) bonded to a primary hydroxyl group(s) of a sulfated polysaccharide, the present inventor has studied intensively a desulfation process in which positional selectivity is high and side reactions such as cleavage of a glycoside bond, elimination of an N-sulfate group(s), etc. are not caused, to accomplish the present invention.
The present invention is a process for producing a desulfated polysaccharide, which comprises reacting a sulfated polysaccharide having a saccharide in which a primary hydroxyl group is sulfated, as a constituent sugar, with a silylating agent represented by the following formula (I) 
wherein R1s are the same or different and each represent a hydrogen atom or a halogen atom, R2 represents a lower alkyl group, and R3s are the same or different and each represent a lower alkyl group, an aryl group or a halogen atom, to selectively desulfate a sulfate group bonded to the primary hydroxyl group.
In the above process, it is preferred that the sulfated polysaccharide is converted into an organic solvent-soluble salt such as an organic basic salt, etc., and the reaction is carried out in an organic solvent.
Also, it is preferred that after the desulfation reaction, a silyl group(s) of a silylated hydroxyl group(s) is/are removed.
The present invention also provides a selectively desulfated heparin which comprises having the following characteristics:
(1) in an unsaturated disaccharide composition measured by a method using enzymatic digestion and high performance liquid chromatography, the xcex94DiHS-tri(U,6,N)S content shown in FIG. 2 is 10 to 40%, and the xcex94DiHS-di(U,N)S content of the same is 30 to 60%,
(2) the content of a disaccharide containing an N-substituted sulfate group(s) is 75 to 95%,
(3) the weight average molecular weight (Mw) is 4,000 to 30,00dalton,
(4) the anti-thrombin activity is 20 U/mg to 150 U/mg, and
(5) the effect of accelerating the activity of a basic fibroblast growth factor to cellular proliferation is maintained to be 80% or more of that of heparin which is not desulfated.
Further, the present invention provides a selectively desulfated heparin obtained by desulfating heparin according to the above production process, and having the above characteristics (1) to (5).
Further, the present invention provides an agent for accelerating the activity of a basic fibroblast growth factor, containing the above desulfated heparin as an active ingredient, and a composition in which the activity of a basic fibroblast growth factor to cellular proliferation is accelerated, containing the above desulfated heparin and a basic fibroblast growth factor.